Benzodioxane-Benzamides as FtsZ Inhibitors: Effects of Linker's Functionalization on Gram-Positive Antimicrobial Activity

Abstract

FtsZ is an essential bacterial protein abundantly studied as a novel and promising target for antimicrobials. FtsZ is highly conserved among bacteria and mycobacteria, and it is crucial for the correct outcome of the cell division process, as it is responsible for the division of the parent bacterial cell into two daughter cells. In recent years, the benzodioxane-benzamide class has emerged as very promising and capable of targeting both Gram-positive and Gram-negative FtsZs. In this study, we explored the effect of including a substituent on the ethylenic linker between the two main moieties on the antimicrobial activity and pharmacokinetic properties. This substitution, in turn, led to the generation of a second stereogenic center, with both erythro and threo isomers isolated, characterized, and evaluated. With this work, we discovered how the hydroxy group slightly affects the antimicrobial activity, while being an important anchor for the exploitation and development of prodrugs, probes, and further derivatives.

Keywords: 1,4-benzodioxane; Bacillus subtilis; Gram-positive-dependent diseases; antimicrobial resistance; benzamide; cell division protein FtsZ; multidrug-resistant Staphylococcus aureus.

Figure 1

Structures of 2,6-difluoro-3-nonyloxybenzamide (left) and PC190723 (right).

Figure 2

Compound FZ14, progenitor of the benzodioxane benzamide FtsZ inhibitors class; compound FZ88, its improved homologue, and compounds FZ95 and FZ100, presently the strongest derivatives of the series.

Figure 3

Structures of compounds FZ104, FZ105, FZ98, FZ97, FZ118 and FZ119, bearing the methyl substituent (highlighted in blue), and of compounds FZ112, FZ113, FZ116 and FZ117, bearing the hydroxy substituent (highlighted in red), which are the focus of the present study.

Scheme 1

Reagents and solvents: (a) NaBH₄, MeOH, 0 °C; (b) methanesulfonyl chloride (MsCl), triethylamine (TEA), dichloromethane (DCM), RT; (c) 2,6-difluoro-3-hydroxybenzamide, K₂CO₃, dimethylformamide (DMF), 80 °C.

Scheme 2

Reagents and solvents: (a) NaHCO₃, NaIO₄, DCM, RT; (b) CH₃MgBr, THF, RT; (c) NaH, THF, BnBr, RT; (d) H₂SO₄ 2M, dioxane, reflux; (e) MsCl, TEA, DCM, RT; (f) catechol, K₂CO₃, acetone, 60 °C; (g) H₂, Pd/C, MeOH; (h) MsCl, TEA, DCM, RT; (i) 2,6-difluoro-3-hydroxybenzamide, K₂CO₃, DMF, 80 °C.

Scheme 3

Reagents and solvents: (a) Methyl triphenylphosphonium bromide, t-BuOK, THF, RT; (b) BH₃ in THF (1M), H₂O, NaOH, H₂O₂, THF, RT; (c) MsCl, TEA, DCM, RT; (d) 2,6-difluoro-3-hydroxybenzamide, K₂CO₃, DMF, 80 °C.

Scheme 4

Reagents and solvents: (a) Diisopropylamine (DIPA), n-butyl lithium, THF, −78 °C; (b) Br₂, DCM, 0 °C; (c) trimethyl orthoformate, H₂SO₄, CH₃OH, reflux; (d) K₂CO₃, DMF, 70 °C; (e) LiAlH₄, THF, RT; (f) MsCl, TEA, DCM, 0 °C; (g) 2,6-difluoro-3-hydroxybenzamide, K₂CO₃, DMF, 70 °C.

Figure 4

Effects of the compounds on viability of B. subtilis strain WM5126, showing spot dilutions of cultures on agar medium (left) and MICs from broth microdilutions (right) in the presence of the more potent (A) and less potent (B) compounds, as compared with the previously characterized FZ100. N.D.: not determined.

Figure 5

Effects of the compounds on FtsZ assembly and patterning in live B. subtilis WM 5126 cells expressing GFP-ZapA as a proxy for FtsZ. Arrows highlight normal FtsZ rings, both in single dividing cells (left panel), chains of dividing cells (left panel), and nondividing filamentous cells (middle and right panels). Forked arrows highlight FtsZ speckles that result from the disruption of FtsZ rings by FZ116 and, to a lesser extent, by the same concentration of the comparatively less potent compound FZ117. Scale bar, 5 μm.

Figure 6

(A) Superposition of crystallographic (grey colored) and docked TXA 707 (yellow colored). The RMSD value is 0.189 Å. (B) Focus on the three key interactions of TXA707 in the binding site.

Figure 7

FZ117 pose (A) and FZ116 pose (B), superimposed with FZ100 (orange coloured). Hydrogen bonds are highlighted in yellow to be clearly visible.